This invention relates to novel chemically modified nucleic acid molecules that are capable of modulating gene expression through a variety of mechanisms. Specifically, the invention concerns novel combinations of chemical modifications in an oligonucleotide which enhance nuclease resistance, binding affinity, and/or potency.
The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
Since the discovery of the mechanisms underlying gene expression, specifically nucleic acid based transcription and translation, a great deal of effort has been placed on blocking or altering these processes for a variety of purposes, such as understanding biology, gene function, disease processes, and identifying novel therapeutic targets. Approaches involving nucleic acid molecules for modulating gene expression have gained popularity in recent years. For example, nucleic acid molecules have been designed which are capable of binding to specific mRNA sequences by Watson-Crick base pairing interaction and blocking translation (Crooke, 1996, Medicinal Res. Rev. 16, 319-344). Another approach involves complexation of DNA with triplex forming oligonucleotides to prevent transcription of bound DNA sequences thereby inhibiting gene expression (Kim et al., 1998, Biochemistry. 37, 2299-2304). The interaction of antisense oligonucleotides, 2-5A antisense chimera, or ribozymes with target RNA have been used to prevent gene expression. All of these nucleic acid molecules are highly specific to their matching target sequences and therefore can offer lower toxicity compared to traditional approaches such as chemotherapy.
The use of oligonucleotides for modulation of gene expression generally requires stabilization of oligonucleotides from degradation by nucleases that are present in biological systems. Cellular efficacy can be effected if the nucleic acid molecule is degraded before it reaches its desired target. Chemical modifications of nucleic acid molecules have been found to be advantageous in making them inaccessible to degradation by cellular nucleases. Uhlmann and Peyman, 1990, Chem. Reviews 90, 543, review the use of nucleoside modifications to stabilize antisense oligonucleotides. Besides improved stability, chemical modifications have also been shown to increase binding affinity, improve cellular penetration, and enhanced target specificity (Monia et al., 1993, J. Biol. Chem. 268, 14514-14522; Wu-Pong, 1994, BioPharm, 22-33).
One of the most studied and utilized chemical alteration in oligonucleotides has been backbone modifications such as phosphorothioates. Phosphorothioate oligonucleotides are nucleic acid molecules whose phosphodiester linkage has been modified by substituting a sulfur atom in place of an oxygen atom. In addition to increased nuclease resistance, phosphorothioate oligonucleotides are substrates for ribonuclease H (RNase H) (Monia, supra; Crooke et al., 1995, Biochem. J. 3112, 599-608). RNase H is an endonuclease which catalyzes the degradation of RNA in an RNA-DNA heteroduplex (Hostomsky et al., 1993 in Nucleases, Linn et al., eds., Cold Spring Harbor Laboratory Press, NY, 341-376). RNA/DNA heteroduplexes, called Okazaki fragments, are formed naturally during DNA replication. Therefore, the normal function of RNase H is to degrade the RNA portion of the heteroduplex to complete DNA replication. In experiments with E. coli RNase H, the phosphorothioate oligonucleotide activated the enzyme more efficiently (2-5 fold) compared to a standard phosphodiester containing oligonucleotide (Crooke, 1995, supra).
Binding of DNA to RNA is not as thermodynamically favorable as an RNA to RNA interaction (Altmann et al., 1996, Chimia 50, 168-176). Inoe and Ohtsuka, 1987, Nucleic Acids Research 115, 6131, first proposed an oligonucleotide with a central region consisting of oligodeoxynucleotides flanked by 2xe2x80x2-O-methyl modified nucleotide regions. The region of oligodeoxynucleotides in such a chimeric molecule is recognized by RNase H when bound to target RNA; and facilitates cleavage of target RNA by RNase H. (Inoe and Ohtsuka, 1987, FEBS Lett. 215, 327; Shibahara and Morisava, 1987, Nucleic Acids Res. 15, 4403). Such chimeric oligonucleotides were proposed to interact with target RNA more stably than an all DNA oligonucleotide.
Subsequent developments included the introduction of nuclease resistant modifications of the chimeric oligonucleotides, such as methylphosphonates (Tidd and Gibson, 1988, Anticancer Drug Design 3, 117), phosphorothioates (Agrawal and Pederson, 1990, Proc Nat. Acad. Sci. USA 87, 1407), and phosphoramidates (Potts and Runyun, 1991, Proc Nat. Acad. Sci. USA 88, 1516). Additionally, the flanking sequences have been modified with 2xe2x80x2-O-methyl and 2xe2x80x2-F-modifications (Cook, 1993, Antisense Research and Applications, CRC Press, 150-181).
Agrawal et al., U.S. Pat. No. 5,652,355, describe a phosphorothioate-containing nucleic acid molecule with at least two 2xe2x80x2-O-methyl modifications on the 5xe2x80x2 and 3xe2x80x2 ends.
Agrawal, U.S. Pat. No. 5,652,356, describes an oligonucleotide which consists of a region of 2xe2x80x2-O-substituted oligonucleotide located between two oligodeoxyribonucleotide regions. The DNA regions of this nucleic acid molecule consists of phosphorothioate modifications at every position.
Cook et al., U.S. Pat. No. 5,623,065, describe the use of a nucleic acid molecule which contains an RNase H cleavable region flanked by certain specifically modified nucleotides, for inhibition of gene expression of a ras gene.
Cook et al., U.S. Pat. No. 5,587,362, describe a nucleic acid molecule having xe2x80x9csubstantially chirally pure inter-sugar linkagesxe2x80x9d, for modulation of gene expression.
Ohtsuka et al., U.S. Pat. No. 5,013,830, describe mixed oligomers having a DNA region and a 2xe2x80x2-O-methyl modified region, useful for modulation of gene expression.
Walder et al., U.S. Pat. No. 5,491,133, describe a method for modulating gene expression using chimeric oligonucleotides with 3xe2x80x2-phosphodiester linkage modifications.
Cohen et al., U.S. Pat. No. 5,276,019, and Cohen et al., U.S. Pat. No. 5,264,423 describe the use of oligodeoxynucleotides of no more than 32 nucleotides in length, containing at least one phosphorothioate internucleoside linkage which are capable of preventing foreign nucleic acid replication.
Cohen et al., U.S. Pat. No. 5,286,717, describe an oligodeoxyribonucleotide with at least one phosphorothioate modification capable of inhibiting oncogenes.
Sproat et al., U.S. Pat. No. 5,334,711, describe 2xe2x80x2-O-R modified hammerhead and hairpin ribozymes, where R is alkyl, alkynyl or alkenyl.
Crooke et al., 1996, Exp. Opin. Ther. Patents 6, 855, list and discuss various patents and PCT publications in the field of antisense technology.
Sproat et al., U.S. Pat. No. 5,678,731, describe 2xe2x80x2-O-R modified oligonucleotides where R is alkyl, alkynyl or alkenyl.
Usman et al., U.S. Pat. No. 5,652,094, describe enzymatic nucleic acid molecules which include nucleic acid analogues or deoxyribonucleotides.
Joyce, International Publication No. WO 96/17086, describes a DNA enzyme capable of cleaving RNA.
Rossi et al., U.S. Pat. No. 5,144,019, describe chimeric hammerhead ribozymes with the binding arms and stem II region modified with deoxyribonucleotides.
Arrow et al., U.S. Pat. Nos. 5,989,912 and 5,849,902, describe three component chimeric antisense oligonucleotides.
Tullis, U.S. Pat. No. 5,919,619, describes methods for inhibiting target protein expression with specific antisense nucleic acid molecules.
Walder et al., U.S. Pat. No. 5,144,019, describe the use of specific oligodeoxynucleotides modified at the 3xe2x80x2-terminal internucleotide link as therapeutic agents by a method of hybridizing the modified oligonucleotide to a complementary sequence within a targeted mRNA and cleaving the mRNA within the RNA-DNA helix by the enzyme RNaseH to block the expression of the corresponding gene.
Crooke et al., U.S. Pat. No. 6,001,653, describe the use of specific antisense oligonucleotides that that bind to a target RNA molecule and cleave the RNA via RNAse H mediated activity.
Molecules have also been devised which include non-nucleotides capable of binding to nucleic acid. These peptide nucleic acid (PNA) molecules bind by Watson-Crick base-pairing and can also function through an antisense mechanism. These molecules have been used to augment hammerhead ribozyme activity by altering the structure of target RNAs and increasing accessibility of cleavage sites (Jankowsky et al., 1997, Nucleic Acids Research 25, 2690-2693).
This invention relates to novel nucleic acid molecules which are useful for modulation of gene expression. The nucleic acid molecule of the instant invention are distinct from other nucleic acid molecules known in the art. Specifically, the nucleic acid molecules of the present invention have novel combinations of chemical modifications and are capable of binding to RNA or DNA to facilitate modulation of gene expression. These novel combinations of chemical modifications can be used to form antisense oligonucleotides, triplex forming oligonucleotides, 2-5A antisense chimera, and enzymatic nucleic acid molecules.
In one embodiment, the invention features a nucleic acid molecule having the following formulae: Formula I:
5xe2x80x2Cxe2x80x94(X)mxe2x80x94(Y)nxe2x80x94(X)oxe2x80x94Cxe2x80x23xe2x80x2
Formula II:
5xe2x80x2Cxe2x80x94(Y)nxe2x80x94(X)rxe2x80x94Cxe2x80x23xe2x80x2
Formula III:
5xe2x80x2Cxe2x80x94(X)rxe2x80x94(Y)nxe2x80x94Cxe2x80x23xe2x80x2
In a another embodiment, the invention features an enzymatic nucleic acid molecule having the formula:
Formula IV:
5xe2x80x2Cxe2x80x94(X)mxe2x80x94(Z)pxe2x80x94(X)oxe2x80x94(Y)nxe2x80x94(X)qxe2x80x94Cxe2x80x23xe2x80x2
Formula V:
5xe2x80x2Cxe2x80x94(X)mxe2x80x94(Y)nxe2x80x94(X)oxe2x80x94(Z)pxe2x80x94(X)qxe2x80x94Cxe2x80x23xe2x80x2
Formula VI:
5xe2x80x2Cxe2x80x94(Y)nxe2x80x94(Z)pxe2x80x94(X)qxe2x80x94Cxe2x80x23xe2x80x2
Formula VII
5xe2x80x2Cxe2x80x94(X)qxe2x80x94(Z)pxe2x80x94(Y)nxe2x80x94Cxe2x80x23xe2x80x2
In each of the above formula (I-VII), X represents independently a nucleotide which can be same or different; where m and o are integers independently greater than or equal to 4 and preferably less than about 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; r is an integer greater than or equal to four, more specifically 5, 6, 7, 10, 15, or 20; the nucleic acid molecule can be symmetric (m equal to O) or asymmetric (m not equal to O); (X)m, (X)o, and (X)q are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid molecule (the target can be an RNA, DNA or RNA/DNA mixed polymers); Y represents independently a deoxyribonucleotide which can be same or different; n is an integer greater than or equal to 4, specifically 5, 6 7, 8, 9, 10, 11, or 12; Z represents an oligonucleotide including nucleotides capable of facilitating the cleavage of a target sequence; p is of length greater than or equal to 4 but less than 100, preferably 5, between 10-20, specifically 25-55, specifically between 30-45, more specifically 35-50; q is an integer greater than or equal to 0, preferably 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20; _represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage or others known in the art); and each (X)m, (X)o, (X)r, (X)q, and/or (Y)n independently comprise phosphorothioate linkages, more specifically each (X)m, (X)o, (X)r, (X)q, and/or (Y)n independently comprise at least one phosphodiester linkage and/or one phosphorothioate linkage or a mixture thereof; each C and Cxe2x80x2 independently represents a cap structure which can independently be present or absent; and (Z)p can optionally include a phosphorothioate linkage. The nucleotides in the each of the formula I-VII are unmodified or modified at the sugar, base, and/or phosphate as known in the art.
In another embodiment, each of X represents independently a nucleotide which can be same or different; where m and o are integers independently greater than or equal to 5; (X)m and (X)o are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid molecule; each (X)r comprises independently at least one phosphodiester linkage and/or one phosphorothioate linkage; Y represents independently a deoxyribonucleotide which can be same or different; (Y)n is an oligonucleotide which is of sufficient length to stably interact independently with a target nucleic acid molecule; n is an integer greater than or equal to 4; each (X)m, and (X)o comprise independently at least one phosphodiester linkage and/or one phosphorothioate linkage or a mixture thereof; (Y)n comprises a phosphorothioate linkage or a phosphorodithioate linkage or a 5xe2x80x2-S-phosphorothioate, or 5xe2x80x2-S-phosphorodithioate, or a 3xe2x80x2xe2x80x94Sxe2x80x94 phosphorothioate or a 3xe2x80x2-S-phosphorodithioate linkage or a mixture thereof; and each C and Cxe2x80x2 independently represents a cap structure which can independently be present or absent.
In one embodiment, Z in the above formulae IV-VII comprises a hammerhead, inozyme, G-cleaver, zinzyme, DNAzyme, or amberzyme enzymatic nucleic acid molecule.
In another embodiment, the invention features a nucleic acid molecule having the following formulae:
XI:
rsrsrsrprprprpDsDsDsDsDsDsDsDsDsrprprprsrsrsD 
XII:
rsrsrsrprprprpDsDsDsDsDsDsDsDsDsrprprprsrsrsr-iB 
XIII:
iB-rprprprprprprpDsDsDsDsDsDsDsDsDsrprprprprprpr-iB 
XIV:
xpxpxpxpxpxpxpDsDsDsDsDsDsDsDsDsxpxpxpxpxpxpx 
In each of the above Formulae (XI-XIV), r stands for any 2xe2x80x2-O-alkyl nucleotide, for example 2xe2x80x2-O-methyl adenosine, 2xe2x80x2-O-methyl guanosine, 2xe2x80x2-O-methyl cytidine, or 2xe2x80x2-O-methyl uridine; D stands for any deoxynucleotide or 2xe2x80x2-arabinofluoro nucleotide, s stands for a phosphorothioate, phosphorodithio ate, 5xe2x80x2-thiophosphate, 3xe2x80x2-thiophosphate or methylphosphonate internucleotide linkage; p stands for a phosphodiester internucleotide linkage; x stands for a 2xe2x80x2-O-alkyl-thio-alkyl nucleotide, for example 2xe2x80x2-methyl-thio-methyl adenosine, 2xe2x80x2-methyl-thio-methyl guanosine, 2xe2x80x2-methyl-thio-methyl cytidine, or 2xe2x80x2-methyl-thio-methyl uridine; and iB stands for a inverted abasic moiety, for example a 3xe2x80x2,3xe2x80x2 or 5xe2x80x2,5xe2x80x2-linked deoxyabasic moiety. The link age between the 3xe2x80x2,3xe2x80x2 or 5xe2x80x2,5xe2x80x2-linked deoxyabasic moiety can be a phosphorothioate, phosphorodithioate, 5xe2x80x2-thiophosphate, 3xe2x80x2-thiophosphate, methylphosphonate, or phosphodiester internucleotide linkage.
In one embodiment, a nucleic acid molecule of the invention is an antisense nucleic acid molecule.
In another embodiment, a nucleic acid molecule of the invention further comprises a 2-5A antisense chimera.
The present invention also features a method of modulating the expression of a gene in a cell, for example a mammalian cell or human cell, comprising the step of administering to the cell a nucleic acid molecule of the invention under conditions suitable for the down regulation of said gene.
In one embodiment, the invention features a pharmaceutical composition comprising a nucleic acid molecule of the invention in a pharmaceutically acceptable carrier.
In another embodiment, the invention features a method of administering to a cell a nucleic acid molecule of the invention, comprising contacting the cell, for example a mammalian cell or human cell, with the nucleic acid molecule under conditions suitable for the administration. The administration can be in the presence of a delivery reagent, for example, a lipid, cationic lipid, or liposome.
The term xe2x80x9cnucleotidexe2x80x9d as used herein, refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1xe2x80x2 position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman and Peyman, supra all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5xe2x80x2-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman and Peyman, supra). By xe2x80x9cmodified basesxe2x80x9d in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1xe2x80x2 position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
The term xe2x80x9c2xe2x80x2-arabinofluoro nucleotidexe2x80x9d as used herein, refers to a nucleotide comprising a 2xe2x80x2-fluoro group in an arabinofuranosyl configuration.
The term xe2x80x9cunmodified nucleotidexe2x80x9d as used herein, refers to a nucleotide with one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1xe2x80x2 carbon of xcex2-D-ribo-furanose.
The term xe2x80x9cmodified nucleotidexe2x80x9d as used herein, refers to a nucleotide which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
The term xe2x80x9csufficient lengthxe2x80x9d as used herein, refers to an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, xe2x80x9csufficient lengthxe2x80x9d means that the binding sequence of an antisense nucleic acid is long enough to provide stable binding to a target site under the expected binding conditions.
The term xe2x80x9cstably interactxe2x80x9d as used herein, refers to the interaction of nucleic acid molecules of the invention with a target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions). The interaction is stable either alone or in conjunction with (Y)n and (Z)p where applicable.
The term xe2x80x9cchimeric nucleic acid moleculexe2x80x9d or xe2x80x9cchimeric oligonucleotidexe2x80x9d as used herein, refers to a nucleic acid molecule that can be comprised of both modified or unmodified DNA or RNA.
The term xe2x80x9ccap structurexe2x80x9d as used herein, refers to chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5xe2x80x2-terminus (5xe2x80x2-cap) or at the 3xe2x80x2-terminus (3xe2x80x2-cap) or can be present on both terminus. In non-limiting examples, the 5xe2x80x2-cap includes inverted abasic residue (moiety), 4xe2x80x2,5xe2x80x2-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3xe2x80x2-3xe2x80x2-inverted nucleotide moiety; 3xe2x80x2-3xe2x80x2-inverted abasic moiety; 3xe2x80x2-2xe2x80x2-inverted nucleotide moiety; 3xe2x80x2-2xe2x80x2-inverted abasic moiety; 1,4-butanediol phosphate; 3xe2x80x2-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3xe2x80x2-phosphate; 3xe2x80x2-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).
In one embodiment the cap structure includes, for example 4xe2x80x2,5xe2x80x2-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 5xe2x80x2-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5xe2x80x2-5xe2x80x2-inverted nucleotide moiety; 5xe2x80x2-5xe2x80x2-inverted abasic moiety; 5xe2x80x2-phosphoramidate; 5xe2x80x2-phosphorothioate; 1,4-butanediol phosphate; 5xe2x80x2-amino; bridging and/or non-bridging 5xe2x80x2-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5xe2x80x2-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
In another embodiment (X)m, (X)o, (X)q, (Y)n and/or (Z)p independently include modifications selected from a group comprising 2xe2x80x2-O-alkyl (e.g. 2xe2x80x2-O-allyl; Sproat et al., supra); 2xe2x80x2-O-alkylthioalkyl (e.g. 2xe2x80x2-O-methylthiomethyl; Karpeisky et al., 1998, Nucleosides and Nucleotides 16, 955-958); L-nucleotides (Tazawa et al., 1970, Biochemistry 3499; Ashley, 1992, J. Am. Chem. Soc. 114, 9731; Klubmann et al., 1996, Nature Biotech 14, 1112); 2xe2x80x2-C-alkyl (Beigelman et al., 1995,J. Biol. Chem. 270, 25702); 1-5-Anhydrohexitol; 2,6-diaminopurine (Strobel et al., 1994, Biochem. 33, 13824-13835); 2xe2x80x2-(N-alanyl) amino-2xe2x80x2-deoxynucleotide; 2xe2x80x2-(N-beta-alanyl) amino; 2xe2x80x2-deoxy-2xe2x80x2-(lysyl) amino; 2xe2x80x2-O-amino (Karpeisky et al., 1995, Tetrahedron Lett. 39, 1131); 2xe2x80x2-deoxy-2xe2x80x2-(N-histidyl) amino; 5-methyl (Strobel, supra); 2xe2x80x2-(N-b-carboxamidine-beta-alanyl) amino; 2xe2x80x2-deoxy-2xe2x80x2-(N-beta-alanyl) (Matulic-Adamic et al., 1995, Bioorg. and Med. Chem. Lett. 5,2721-2724); xylofuranosyl (Rosemeyer et al., 1991, Helvetica Chem. Acta, 74, 748; Seela et al., 1994, Helvetica Chem. Acta, 77, 883; Seela et al., 1996, Helvetica Chem. Acta, 79, 1451).
In another embodiment, the invention features a nucleic acid molecule of any of formula I-VII, where each X and/or Z, independently include a nucleotide modification having formula IX: 
Wherein, each B is independently a modified or an unmodified nucleic acid base; R1 is independently a fluoroalkyl or an alkylthiofluoroalkyl; E is independently a phosphorus-containing group; and D is independently an O, blocking group or a phosphorus-containing group.
In another embodiment, the invention features a nucleic acid molecule of any of formula I-VII, where each X and/or Z, independently include a nucleotide modification having formula X: 
Wherein, each B is independently a modified or an unmodified nucleic acid base; R1 is independently an aklyl, an alkylthioalkyl, a fluoroalkyl or an alkylthiofluoroalkyl; E is independently a phosphorus-containing group; and D is independently an O, blocking group or a phosphorus-containing group.
The term xe2x80x9calkylxe2x80x9d as used herein refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain xe2x80x9cisoalkylxe2x80x9d, and cyclic alkyl groups. The term xe2x80x9calkylxe2x80x9d also comprises alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from about 1 to 7 carbons, more preferably about 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. The term xe2x80x9calkylxe2x80x9d also includes alkenyl groups containing at least one carbonxe2x80x94carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has about 2 to 12 carbons. More preferably it is a lower alkenyl of from about 2 to 7 carbons, more preferably about 2 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. The term xe2x80x9calkylxe2x80x9d also includes alkynyl groups containing at least one carbonxe2x80x94carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has about 2 to 12 carbons. More preferably it is a lower alkynyl of from about 2 to 7 carbons, more preferably about 2 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moieties of the invention can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An xe2x80x9calkylarylxe2x80x9d group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from about 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An xe2x80x9camidexe2x80x9d refers to an xe2x80x94C(O)xe2x80x94NHxe2x80x94R, where R is either alkyl, aryl, alkylaryl or hydrogen. An xe2x80x9cesterxe2x80x9d refers to an xe2x80x94C(O)xe2x80x94ORxe2x80x2, where R is either alkyl, aryl, alkylaryl or hydrogen.
The term xe2x80x9calkoxyalkylxe2x80x9d as used herein refers to an alkyl-O-alkyl ether, for example methoxyethyl or ethoxymethyl.
The term xe2x80x9calkyl-thio-alkylxe2x80x9d as used herein refers to an alkyl-S-alkyl thioether, for example methylthiomethyl or methylthioethyl.
The term xe2x80x9caminoxe2x80x9d as used herein refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms xe2x80x9caminoacylxe2x80x9d and xe2x80x9caminoalkylxe2x80x9d refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
The term xe2x80x9caminationxe2x80x9d as used herein refers to a process in which an amino group or substituted amine is introduced into an organic molecule.
The term xe2x80x9cexocyclic amine protecting moietyxe2x80x9d as used herein refers to a nucleobase amino protecting group compatible with oligonucleotide synthesis, for example an acyl or amide group.
The term xe2x80x9calkenylxe2x80x9d as used herein refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbonxe2x80x94carbon double bond. Examples of xe2x80x9calkenylxe2x80x9d include vinyl, allyl, and 2-methyl-3-heptene.
The term xe2x80x9calkoxyxe2x80x9d as used herein refers to an alkyl group of indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.
The term xe2x80x9calkynylxe2x80x9d as used herein refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbonxe2x80x94carbon triple bond. Examples of xe2x80x9calkynylxe2x80x9d include propargyl, propyne, and 3-hexyne.
The term xe2x80x9carylxe2x80x9d as used herein refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring can optionally be fused or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene and biphenyl. Preferred examples of aryl groups include phenyl and naphthyl.
The term xe2x80x9ccycloalkenylxe2x80x9d as used herein refers to a C3-C8 cyclic hydrocarbon containing at least one carbonxe2x80x94carbon double bond. Examples of cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
The term xe2x80x9ccycloalkylxe2x80x9d as used herein refers to a C3-C8 cyclic hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
The term xe2x80x9ccycloalkylalkyl,xe2x80x9d as used herein, refers to a C3-C7 cycloalkyl group attached to the parent molecular moiety through an alkyl group, as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
The terms xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as used herein refers to indicate fluorine, chlorine, bromine, and iodine.
The term xe2x80x9cheterocycloalkyl,xe2x80x9d as used herein refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused to or otherwise attached to other heterocycloalkyl rings and/or non-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups have from 3 to 7 members. Examples of heterocycloalkyl groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, and pyrolidinyl.
The term xe2x80x9cheteroarylxe2x80x9d as used herein refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings or heterocycloalkyl rings. Examples of heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples of heteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.
The term xe2x80x9cC1-C6 hydrocarbylxe2x80x9d as used herein refers to straight, branched, or cyclic alkyl groups having 1-6 carbon atoms, optionally containing one or more carbonxe2x80x94carbon double or triple bonds. Examples of hydrocarbyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene, cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl and propargyl. When reference is made herein to C1-C6 hydrocarbyl containing one or two double or triple bonds it is understood that at least two carbons are present in the alkyl for one double or triple bond, and at least four carbons for two double or triple bonds.
A xe2x80x9cblocking groupxe2x80x9d is a group which is able to be removed after polynucleotide synthesis and/or which is compatible with solid phase polynucleotide synthesis.
A xe2x80x9cphosphorus containing groupxe2x80x9d can include phosphorus in forms such as dithioates, phosphoramidites and/or as part of an oligonucleotide.
The term xe2x80x9cabasicxe2x80x9d as used herein, refers to moieties lacking a base or having other chemical groups in place of a base at the 1xe2x80x2 position, for example a 3xe2x80x2,3xe2x80x2-linked or 5xe2x80x2,5xe2x80x2-linked deoxyabasic ribose derivative (for more details see Wincott et al., International PCT publication No. WO 97/26270).
In one embodiment Cxe2x80x2 is selected from a group comprising inverted abasic residue, 4xe2x80x2,5xe2x80x2-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4xe2x80x2-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3xe2x80x2-3xe2x80x2-inverted nucleotide moiety; 3xe2x80x2-3xe2x80x2-inverted abasic moiety; 3xe2x80x2-2xe2x80x2-inverted nucleotide moiety; 3xe2x80x2-2xe2x80x2-inverted abasic moiety; 1,4-butanediol phosphate; 3xe2x80x2-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3xe2x80x2-phosphate; 3xe2x80x2-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Beigelman et al., International PCT publication No. WO 97/26270, incorporated by reference herein).
In another embodiment C is selected from a group comprising, 4xe2x80x2,5xe2x80x2-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 5xe2x80x2-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5xe2x80x2-5xe2x80x2-inverted nucleotide moeity; 5xe2x80x2-5xe2x80x2-inverted abasic moeity; 5xe2x80x2-phosphoramidate; 5xe2x80x2-phosphorothioate; 1,4-butanediol phosphate; 5xe2x80x2-amino; bridging and/or non-bridging 5xe2x80x2-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5xe2x80x2-mercapto moeities (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
In another embodiment (Z)p includes a non-nucleotide linker. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule. Non-nucleotides as can include abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides and Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A xe2x80x9cnon-nucleotidexe2x80x9d further means any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.
The term xe2x80x9cenzymatic nucleic acid moleculexe2x80x9d as used herein, refers to a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions (e.g. (X)m, (X)o, (X)q and (Y)n in formulae IV-VII) allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids can be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
The term xe2x80x9ccomplementarityxe2x80x9d as used herein, refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). xe2x80x9cPerfectly complementaryxe2x80x9d means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein, refers to a molecule comprising two or more nucleotides.
The term xe2x80x9cenzymatic portionxe2x80x9d as used herein, refers to that part of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate.
The terms xe2x80x9csubstrate binding regionxe2x80x9d, xe2x80x9csubstrate binding armxe2x80x9d or xe2x80x9csubstrate binding domainxe2x80x9d as used herein, refers to that portion/region of a nucleic acid, for example an antisense nucleic acid or enzymatic nucleic acid molecule, which is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate. Preferably, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in FIGS. 1-4. That is, these arms contain sequences within a enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions. The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths. The length of the binding arm(s) are preferably greater than or equal to three nucleotides and of sufficient length to stably interact with the target RNA; preferably 12-100 nucleotides; more preferably 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranz et al., 1993, EMBO J., 12, 2567-73). If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
The term xe2x80x9cRNAxe2x80x9d as used herein, refers to a molecule comprising at least one ribonucleotide residue. By xe2x80x9cribonucleotidexe2x80x9d or xe2x80x9c2xe2x80x2-OHxe2x80x9d is meant a nucleotide with a hydroxyl group at the 2xe2x80x2 position of a xcex2-D-ribo-furanose moiety.
The term xe2x80x9cInozymexe2x80x9d or xe2x80x9cNCHxe2x80x9d motif or configuration as used herein, refers to an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG. 1. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and / represents the cleavage site. H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site. xe2x80x9cIxe2x80x9d in FIG. 1 represents an Inosine nucleotide, preferably a ribo-Ino sine or xylo-Ino sine nucleoside.
The term xe2x80x9cG-cleaverxe2x80x9d motif or configuration as used herein, refers to an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site. G-cleavers can be chemically modified as is generally shown in FIG. 1.
The term xe2x80x9camberzymexe2x80x9d motif or configuration as used herein, refers to an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 2. Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site. Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 2. In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5xe2x80x2-gaaa-3xe2x80x2 loops shown in the figure. Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2xe2x80x2-OH) group within its own nucleic acid sequence for activity.
The term xe2x80x9czinzymexe2x80x9d motif or configuration as used herein, refers to an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 3. Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site. Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 3, including substituting 2xe2x80x2-O-methyl guanosine nucleotides for guanosine nucleotides. In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5xe2x80x2-gaaa-2xe2x80x2 loop shown in the figure. Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2xe2x80x2-OH) group within its own nucleic acid sequence for activity.
By xe2x80x98DNAzymexe2x80x99 is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2xe2x80x2-OH group within its own nucleic acid sequence for activity. In particular embodiments the enzymatic nucleic acid molecule can have an attached linker(s) or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2xe2x80x2xe2x80x94OH groups. DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in FIG. 4 and is generally reviewed in Usman et al., U.S. Pat. No., 6,159,714; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am. Chem. Soc., 122, 2433-39. Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.
The term xe2x80x9cnucleic acid moleculexe2x80x9d as used herein, refers to a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
In one embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will recognize that these molecules can be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
In another embodiment, the nucleic acid molecule of the present invention can form structures including but not limited to antisense, triplexes, 2-5A chimera antisense, or enzymatic nucleic acid (ribozymes).
The term xe2x80x9cantisense nucleic acidxe2x80x9d, as used herein, refers to a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNAxe2x80x94RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J Biol. Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
The term xe2x80x9cRNase H activating regionxe2x80x9d as used herein, refers to a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; more specifically, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5xe2x80x2-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.
The term xe2x80x9c2-5A antisense chimeraxe2x80x9d as used herein, refers to an antisense oligonucleotide containing a 5xe2x80x2-phosphorylated 2xe2x80x2-5xe2x80x2-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).
The term xe2x80x9ctriplex forming oligonucleotidesxe2x80x9d or xe2x80x9ctriplex DNAxe2x80x9d as used herein, refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181-206).
The term xe2x80x9cgenexe2x80x9d as used herein, refers to a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.
In another embodiment, the invention features an antisense oligonucleotide which is capable of interacting with the target RNA and sterically blocking translation, where the oligonucleotide has a 5xe2x80x2 and a 3xe2x80x2 Cap structure and the oligonucleotide can include modifications at the base, sugar or the phosphate groups.
The nucleic acid molecules of the instant invention are also referred to as GeneBloc reagents, which are essentially nucleic acid molecules (eg; ribozymes, antisense) capable of down-regulating gene expression.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.